An efficiently operating haemostatic system is of vital necessity for the mammalian organism. In healthy organisms, defects of the blood vascular system, e.g. vascular lesions, are repaired in a two-step process: the aggregation of thrombocytes is followed by the formation of a fibrin clot in an enzyme cascade under participation of several blood clotting factors. Most of these factors are serin proteases, for example thrombin which catalyzes the reaction of fibrinogen to fibrin. The coagulation system is counteracted by the fibrinolytic system involving, among others, the protease plasmin which cleaves fibrin. The fibrinolytic system is equally important as the coagulation system since even under normal physiological conditions small amounts of fibrin are formed in the blood and therefore intravascular thrombi would be formed without constant fibrinolysis. Furthermore, the fibrinolytic system is necessary in keeping tubular systems, such as glandular ducts and the efferent urinary tract, free from fibrin precipitates and in dissolving fibrin clots after the structural integrity of a damaged area is restored. The coagulation and fibrinolytic systems are usually in a dynamic equilibrium. In cases, however, in which the fibrinolytic potential of the organism is disturbed or insufficient, for example in patients suffering from tbromboembolisms or post-operative complications, it is indispensable to support the organism by the administration of anticoagulants to prevent further formation of fibrin and of thrombolytic agents to dissolve the formed thrombi.
Hirudin, an anticoagulant that occurs naturally in leeches (Hirudo medicinalis), has been known for a long time. Hirudin is not a single polypeptide species but a class of equally acting polypeptides consisting of at least four representatives designated hirudin variant 1 (HV1), hirudin variant 2 (HV2; EP Application 0 158 564), hirudin variant PA (HV3; PCT Application WO 88/03493), and "des-(Val).sub.2 -hirudin" (EP Application 0 158 986). The variants differ from each other by a number of amino acids, for example at the N-terminal sequence which is Val-Val-Tyr for HV1, Ile-Thr-Tyr for HV2 and PA and Tbr-Tyr for "des-(Val).sub.2 -hirudin". Based on NMR studies, HV1 is composed of an N-terminal core domain with a protruding "finger" (residues 31-36), and an acidic terminal loop (Clore et al., EMBO Journal 6, 529, 1987). All above-mentioned hirudin variants have an accumulation of hydrophobic amino acids at the N-terminus and an accumulation of polar amino acids at the C-terminus, a tyrosine residue (Tyr 63) present as sulphate monoester, three disulphide bridges and the anticoagulant activity in common.
Of all naturally occurring and synthetic anticoagulants which are specific for thrombin, hirudin has the highest affinity for the target enzyme. The inhibitor forms an extremely stable one-to-one molar complex with thrombin which is enzymatically totally inactive. Other enzymes of the coagualation cascade are not inhibited by hirudin.
Hirudin shows promising pharmacokinetic and pharmacodynamic properties (see for example Markwardt et al., Thromb. Haemostasis 47, 226, 1982). No effects on heart rate, respiration, blood pressure, thrombocyte count, fibrinogen and haemoglobin were observed after intravenous administration of hirudin to dogs, even in high doses. In tests on rats, pigs and dogs, hirudin has proved effective in experimental thrombosis, in endotoxin shock, and also in disseminated intravascular coagulation.
One prerequisite for the therapeutic application of hirudin is the possibility to produce sufficient amounts using modern methods of biotechnology. Recently, cDNAs and synthetic genes coding for hirudin variants have been cloned and expressed in microbial hosts. Although the expression products lack the sulphate monoester group at Tyr 63 and were therefore designated desulphatohirudins, they turned out to exhibit biological properties at least equivalent to those of natural sulphated hirudins. Desulphatohirudin variant HV1 bas been expressed in Escherichia coli (EP Applications 0 158 564 and 0 168 342) and in Saccharomyces cerevisiae (EP Applications 0 168 342, 0 200 655, 0 225 633 and 0 252 854). Similarly, desulphatohirudin HV2 has been expressed in E. coli (EP Application 0 200 655, PCT Application WO 86/01224), and des-(Val).sub.2 -desulphatohirudin has been expressed in E. coli (EP Application 0 158 986).
Equally important for future routine administration of the clotting inhibitor is the development of methods for sensitive and reproducible quantitation of the anticoagulant in biological fluids to be able to monitor the drug. Usually, hirudin is assessed via its interaction with thrombin. Due to the fact that hirudin is a very poor immunogen, it has hitherto been problematic to produce antibodies against hirudin which could be used in immunoassays for the determination of the anticoagulant. The European Patent Application 0 168 342 claims monoclonal antibodies specific for hirudin. However, the specification of the application does not contain a characterization of the claimed antibodies which are supposedly elicited against unmodified hirudin. In view of the poor immunogenic properties of hirudin, the conceptual approach to the production of anti-hirudin monoclonal antibodies in the above-cited patent application lacks the basis for a successful immunization procedure and thus, the successful production of such antibodies. Spinner et al. (J. Immunol. Methods 87, 79, 1986) describe the production of polyclonal anti-hirudin antibodies (antisera) by immunization of sheep with hirudin. The article expressly notes the limited success of the immunization procedure and the difficulties in producing antisera at all. The same research group describes three monoclonal antibodies to hirudin (Stoffler et al., Thrombosis Res. Suppl. 7, 38, 1987), one of which interferes with the interaction of hirudin with .alpha.-thrombin. However, neither the immunization procedure, especially the hirudin variant used as antigen, nor the interaction between the monoclonal antibody and the hirudin/.alpha.-thrombin complex are further characterized in the communication.